Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

Flow Cyt

Use 1µg for 106 cells.

ICC/IF

Use a concentration of 5 - 10 µg/ml.

IHC-FoFr

Use at an assay dependent concentration.

WB

Use a concentration of 5 - 10 µg/ml. Detects a band of approximately 300 kDa (predicted molecular weight: 300 kDa). Abcam recommends using BSA as the blocking agent.

IP

Use at an assay dependent concentration.

Target

FunctionTransport of phosphorylated lysosomal enzymes from the Golgi complex and the cell surface to lysosomes. Lysosomal enzymes bearing phosphomannosyl residues bind specifically to mannose-6-phosphate receptors in the Golgi apparatus and the resulting receptor-ligand complex is transported to an acidic prelyosomal compartment where the low pH mediates the dissociation of the complex. This receptor also binds IGF2. Acts as a positive regulator of T-cell coactivation, by binding DPP4.

DomainContains 15 repeating units of approximately 147 AA harboring four disulfide bonds each. The most highly conserved region within the repeat consists of a stretch of 13 AA that contains cysteines at both ends.

Anti-M6PR (cation independent) antibody [2G11] images

ICC/IF image of ab2733 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab2733, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunofluorescent imaging of human cells (U2OS) with ab2733 confirms the specificity of this antibody, with the expected perinuclear vesicular staining of lysosomes.

IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes. All blocking and incubation steps carried out at 37 degrees.

ab2733 positively staining formaldehyde fixed Human HEK 293 cells (red) in conjunction with goat anti mouse (Alexa 546). Nuclear staining was obtained using Hoechst. This image is an edited version of an image received courtesy of an Abreview submitted by Kun Liu on 19 September 2005. We do not have any further information relating to this image.

ICC/IF image of ab2733 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2733, 10µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Thank you, I received the data this time. For a protocol modification, I suggest trying preparing a sample by heating at 60C for 10 minutes instead of 95 for 5 minutes to minimize aggregation of the trans-membrane domains. It is strange that both antib...

Thank you for contacting us regarding ab2733.
I have confirmed with the laboratory that this particular antibody is only batch tested in IF, but has been tested in WB. I have attached an image for you.
We have recently done some optimisation ...

I'm glad that you have been seeing better results with blocking with BSA. If you are now having difficulty with non-specific bands, increasing the dilution of the primary and secondary antibody as well as adding 1 % BSA and 0.2% Tween 20 into the dilue...

Thank you for your reply.
I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for ab8093.
To check the status of the order please...

Thank you for your reply.
As this antibody, ab2733 is a monoclonal antibody I would not expect much lot to lot variation. It is possible that there was something wrong with just this particular vial as we have not received reports of other problem...